Project II. piRNA pathway organization and precursor processing William Theurkauf, P. I. Project Summary PIWI interacting RNAs (piRNAs) have a conserved function in transcriptional and post- transcriptional transposon silencing in the germline, which is dedicated to transmitting the inherited genome. Here we focus on identifying conserved organizational principles and molecular mechanisms driving transposon control by the piRNA pathway. Transposon control by piRNAs is best understood in Drosophila, where the primary piRNAs that initiate silencing are derived from large heterochromatic ?clusters? composed of nested transposon fragments. Cluster transcripts appear to be processed into mature piRNAs in nuage, which is an electron dense structure closely associated with the cytoplasmic surface of germline nuclei. In flies, heterochromatic clusters at the nuclear periphery appear to organize the perinuclear nuage, forming a compartment that spans the nuclear envelope. This compartment appears to increase the efficiency and specificity of piRNA production, with is critical to germline development. Studies under Aim1 will define this novel germline specific compartment in flies, mouse and worm. How cluster transcripts are differentiated from mRNAs and processed into mature piRNAs is a critical to the specificity of the system, but remains poorly understood. A complex of Drosophila nuclear proteins suppresses cluster transcript splicing, stalled splicing appears to trigger transposon silencing siRNA production in the pathogenic yeast Cryptocccus, and several putative splicing factors are required for fertility in mouse and humans. These observations suggest that stalled splicing has a conserved role in differentiating piRNA precursors from pre-mRNAs. Studies under Aim 2 will broadly define precursor flow though the piRNA pathway, and test the novel hypothesis that regulated splicing differentiates transposons from protein coding. Relevance Transposons and transposon remnants comprise approximately half of the human genome and represent a potentially explosive source of genome instability. Mobilization of these elements can induce insertions, deletions and rearrangements that cause disease, sterility, and developmental defects. The goals of the proposed studies are to define conserved organizational principles and molecular mechanisms that directly impact human reproductive heath.